转氨酶的固定化及酶学性质研究
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摘要
利用环氧树脂载体进行固定化转氨酶的研究,通过单因素优化确定了较优的酶固定化条件:ES-103b树脂载体、转氨酶和辅酶磷酸吡哆醛最佳质量比为50∶6∶5,在p H为7.0的1mol/L磷酸钾缓冲液介质中吸附25 h。将得到的固定化颗粒重新置于pH为9.0的100 mmol/L磷酸钾缓冲液中,30℃保温30 h。取出后置于pH为8.5的3 mol/L甘氨酸-NaOH缓冲液中,25℃保温12 h,制得固定化转氨酶,其比活力为52.7 U/g(湿载体),酶活回收率达到49.3%。酶学性质研究表明:固定化转氨酶催化反应最适pH、温度分别为7.0、50℃;固定化酶热稳定性及p H稳定性明显提高,50℃条件下的半衰期为20.2 d;在pH 8.0的三乙醇胺缓冲液中(4℃),10 d后酶活仍保持初始酶活的82.3%。
This study foc used on the transaminase immobilization using epoxy-activated resin as support. Optimal conditions for immobilization were as follows: Resin ES-103 b, lyophilized enzyme and PLP were added into 1 mol/L potassium phosphate solution(pH 7.0) at the ratio of 50∶6∶5 and incubated at room temperature for 25 h. The obtained immobilized enzyme was placed into 100mmol/L potassium phosphate buffer(p H 9.0) for 30 h at 30 ℃, followed by its incubation in 3 mol/L Glycine-NaOH buffer(pH 8.5) at 25 ℃ for 12 h. The activity of immobilized enzyme was determined as 52.7 U/g(wet support) with activity recovery of 49.3%. The enzymatic characteristics of the immobilized transaminase were investigated. The optimal reaction temperature and pH were 50℃ and 8.0, respectively. The immobilized transaminase possessed an improved p H and thermal stability. The half-time of the immobilized enzyme at 50 ℃ was 20.2 d. The immobilized transaminase remained 82.3% of the original activity after 10 d of storage in TEA buffer(pH 8.0) at 4 ℃.
This study foc used on the transaminase immobilization using epoxy-activated resin as support. Optimal conditions for immobilization were as follows: Resin ES-103 b, lyophilized enzyme and PLP were added into 1 mol/L potassium phosphate solution(pH 7.0) at the ratio of 50∶6∶5 and incubated at room temperature for 25 h. The obtained immobilized enzyme was placed into 100mmol/L potassium phosphate buffer(p H 9.0) for 30 h at 30 ℃, followed by its incubation in 3 mol/L Glycine-NaOH buffer(pH 8.5) at 25 ℃ for 12 h. The activity of immobilized enzyme was determined as 52.7 U/g(wet support) with activity recovery of 49.3%. The enzymatic characteristics of the immobilized transaminase were investigated. The optimal reaction temperature and pH were 50℃ and 8.0, respectively. The immobilized transaminase possessed an improved p H and thermal stability. The half-time of the immobilized enzyme at 50 ℃ was 20.2 d. The immobilized transaminase remained 82.3% of the original activity after 10 d of storage in TEA buffer(pH 8.0) at 4 ℃.
引文
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[8]崔培梧,黎继烈,肖作为,等.环氧基树脂固定化青霉菌柚苷酶的工艺条件[J].食品与发酵工业,2014,40(5):87-92.
[9]丁成勇,徐晓滢,马,等.Eupergit C250L固定化D-海因酶及其催化性质[J].生物加工过程,2007,4(4):41-45.
[2]王潇莹,刘冰滢,游松.转氨酶在手性化合物合成中的研究进展[J].沈阳药科大学学报,2014,31(4):313-324.
[3]SAVILE C K,JANEY J M,MUNDORFF E C,et al.Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture[J].Science,2010,329(5989):305-309.
[4]BRENA B M,BATISTA-VIERA F.Immobilization of enzymes and cells[M].Totowa,NJ:Humana Press,2006:15-30.
[5]TRUPPO M D,STROTMAN H,HUGHES G.Development of an immobilized transaminase capable of operating in organic solvent[J].Chem Cat Chem,2012,4(8):1071-1074.
[6]TORRES P,BATISTA-VIERA F.Immobilization ofβ-galactosidase from Bacillus circulans onto epoxy-activated acrylic supports[J].Journal of molecular catalysis B:Enzymatic,2012,74(3):230-235.
[7]CAO L.Immobilised enzymes:science or art?[J].Current opinion in chemical biology,2005,9(2):217-226.
[8]崔培梧,黎继烈,肖作为,等.环氧基树脂固定化青霉菌柚苷酶的工艺条件[J].食品与发酵工业,2014,40(5):87-92.
[9]丁成勇,徐晓滢,马,等.Eupergit C250L固定化D-海因酶及其催化性质[J].生物加工过程,2007,4(4):41-45.